Selective Kinase Research Articles

Tetrahydropyridine LIMK inhibitors: Structure activity studies and biological characterization

LIM Kinases, LIMK1 and LIMK2, have become promising targets for the development of inhibitors with potential application for the treatment of several major diseases. LIMKs play crucial roles in cytoskeleton remodeling as downstream effectors of small G proteins of the Rho-GTPase family, and as major regulators of cofilin, an actin depolymerizing factor. In this article we describe the conception, synthesis, and biological evaluation of novel tetrahydropyridine pyrrolopyrimidine LIMK inhibitors. Homology models were first constructed to better understand the binding mode of our preliminary compounds and to explain differences in biological activity. A library of over 60 products was generated and in vitro enzymatic activities were measured in the mid to low nanomolar range. The most promising derivatives were then evaluated in cell on cofilin phosphorylation inhibition which led to the identification of 52 which showed excellent selectivity for LIMKs in a kinase selectivity panel. We also demonstrated that 52 affected the cell cytoskeleton by disturbing actin filaments. Cell migration studies with this derivative using three different cell lines displayed a significant effect on cell motility. Finally, the crystal structure of the kinase domain of LIMK2 complexed with 52 was solved, greatly improving our understanding of the interaction between 52 and LIMK2 active site. The reported data represent a basis for the development of more efficient LIMK inhibitors for future in vivo preclinical validation.

Abstract LB161: Discovery of an oral, potent, and selective CDK9 molecular glue degrader SLX-3065 active in aggressive variant prostate cancers (AVPC)

Abstract Background: The use of new potent targeted therapies in prostate cancers has led to the resistance and development of highly aggressive variant prostate cancers (AVPC), including neuroendocrine prostate cancers (NEPC). N-MYC and C-MYC oncogenes overexpress in all aggressive prostate cancers (PCa). This overexpression is highly prevalent in these lethal subtypes of cancers, detected in 2% of all Prostate Cancers and over 10-17% of mCRPC patients. Transcriptional targets of CDK9 are MYCs, which act as an oncogenic driver, sufficient to transform human-derived prostate cancer cells to take on AVPC and NEPC phenotypic changes. Additionally, N-MYC and C-MYC play key roles in tumor drug resistance, and their downregulation, through CDK9 inhibition or degradation lowers tumor growth. Our efforts have demonstrated that targeting drivers of NEPC and mCRPC with highly selective CDK9 molecular glue degraders SLX-3064 SLX-3065 was designed from reversible SLX-3039 for therapeutic intervention of mCRPC, AVPC, and NEPCs. Materials and Methods: We employed our MolecuLernTM-trained molecular glue library design strategies and in vitro CDK9 kinase binding assays. Other experiments include cellular efficacies of single-agent Molecular Glue degraders and their effect in combination experiments on 22Rv1, LASCPC-01, C4-2, and C4-2B MYC expressed PCa cells. Kinase selectivity, Nano BRET, ADME-Tox, in vitro safety, secondary pharmacological assays, Cell engraftment mouse tumor mode, and PK studies were performed. Results: We designed and synthesized a series of novel small molecule CDK9 Molecular Glue degraders based on our initial CDK9 inhibitor lead SLX-3030 and SLX-3039. The SLX-3064 and SLX-3065 reversibly bind to CDK9 with an IC50 of 9 and 7 nM in kinase binding, in target engagement assays, elicit pharmacological responses in N-MYC, C-MYC, driven prostate cancer cells in vitro in LASCPC-01 (21, 35 nM), 22Rv1 (108, 236 nM), C4-2B (15, 12 nM), C4-2 (2.7, 1.5 nM) and VCap (86, 73 nM) cell lines. SLX-3064 and SLX-3065 exhibited potent activity degrading total, pCDK9, MYCs, Mcl-1, and its downstream RNA Pol II, Ser 2, 5, 7, and pAR Ser-81 in a dose-dependent manner. Additionally, we performed in vitro ADME-Tox, hERG, P450, safety panel screen, and in vivo PK experiments in rodent and beagle dog species. Conclusion: In summary, both SLX-3064 and SLX-3065 molecular glues display complete degradation of CDK9 and its downstream markers, and these degraders possess ideal developable criteria as oral agents. With these characteristics, SLX-3065 was selected as a candidate that had the appropriate developable properties and our plans for conducting additional IND-enabling studies and future clinical trial plans will also be discussed. Citation Format: Chenyu Lin, Zhaoliang Li, Kyle Medley, David J. Bearss, Hariprasad Vankayalapati. Discovery of an oral, potent, and selective CDK9 molecular glue degrader SLX-3065 active in aggressive variant prostate cancers (AVPC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB161.

Abstract CT195: First-in-human phase 1 study of WEE1 inhibitor APR-1051 in patients with advanced solid tumors harboring cancer-associated gene alterations

Abstract Background: WEE1 tyrosine kinase plays a key role in cell cycle regulation and DNA damage responses, acting as a principal gatekeeper of the G1-S and G2-M cell cycle checkpoints. Thus, inhibition of WEE1 can result in high cyclin-dependent kinase 1 and 2 activities, leading to aberrant cell cycle progression. Increasing premature entry into mitosis and subsequent apoptosis via WEE1 inhibition is one potential approach to killing cancer cells effectively. APR-1051 is an orally bioavailable, highly potent, small molecule inhibitor of WEE1 that has demonstrated in vivo anti-proliferative activity in multiple cancer cell lines and high selectivity for WEE1 versus polo-like kinases 1, 2, and 3. Increased selectivity for WEE1 may reduce the myelosuppressive toxicity (e.g., limiting Grade 3 toxicities) reported with other WEE1 inhibitors. Methods: The primary aim of this multi-center, open-label Phase I study is to characterize the safety, dose-limiting toxicities (DLTs), maximum tolerated dose (MTD)/maximum administered dose (MAD) and recommended Phase 2 dose (RP2D) of APR-1051 in patients with advanced solid tumors harboring cancer-associated gene alterations. Secondary objectives include evaluating the pharmacokinetic (PK) properties of APR-1051, assessing the effect of food on its PK, and its preliminary efficacy per RECIST version 1.1. The exploratory objective is to assess the pharmacodynamic effects of APR-1051. For dose escalation, an accelerated titration followed by a BOIN design will be utilized to determine the MAD/MTD. Upon completion of the dose escalation, two suitable doses and schedules of single-agent APR-1051 will be determined for further investigation in dose selection optimization. Key inclusion criteria are age at least 18 years with advanced solid tumor and cancer-associated gene alterations (e.g., amplification/overexpression of CCNE1 or CCNE2, deleterious mutations in FBXW7 or PPP2R1A), ECOG PS 0 or 1, and adequate organ function. Key exclusion criteria are cancer therapy within 3 weeks or at least 5 half-lives, prior radiation therapy at the target lesion except if evidence of disease progression, unresolved therapy-related AEs, investigational agent within 5 half-lives or 30 days of study drug, prior WEE1 inhibitor, CNS metastases or unstable involvement, secondary malignancies that are active and/or require therapy, and concomitant moderate/strong inhibitors/inducers of CYP3A4/5, MDR1, or BCRP. Up to 79 patients will be enrolled at 3 to 5 sites in the United States. Citation Format: Timothy A. Yap, Crystal Miller, David Stenehjem, Eric J. Brown, Mike Carleton, Nadeem Q. Mirza. First-in-human phase 1 study of WEE1 inhibitor APR-1051 in patients with advanced solid tumors harboring cancer-associated gene alterations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT195.

High throughput screening identifies dasatinib as synergistic with trametinib in low grade serous ovarian carcinoma

BackgroundLow grade serous ovarian carcinoma (LGSOC) is a distinct histotype of ovarian cancer characterised high levels of intrinsic chemoresistance, highlighting the urgent need for new treatments. High throughput screening in clinically-informative cell-based models represents an attractive strategy for identifying candidate treatment options for prioritisation in clinical studies. MethodsWe performed a high throughput drug screen of 1610 agents across a panel of 6 LGSOC cell lines (3 RAS/RAF-mutant, 3 RAS/RAF-wildtype) to identify novel candidate therapeutic approaches. Validation comprised dose-response analysis across 9 LGSOC models and 5 high grade serous comparator lines. Results16 hits of 1610 screened compounds were prioritised for validation based on >50% reduction in nuclei counts in over half of screened cell lines at 1000 nM concentration. 11 compounds passed validation, and the four agents of greatest interest (dasatinib, tyrosine kinase inhibitor; disulfiram, aldehyde dehydrogenase inhibitor; carfilzomib, proteasome inhibitor; romidepsin, histone deacetylase inhibitor) underwent synergy profiling with the recently approved MEK inhibitor trametinib. Disulfiram demonstrated excellent selectivity for LGSOC versus high grade serous ovarian carcinoma comparator lines (P = 0.003 for IC50 comparison), while the tyrosine kinase inhibitor dasatinib demonstrated favourable synergy with trametinib across multiple LGSOC models (maximum zero interaction potency synergy score 46.9). The novel, highly selective Src family kinase (SFK) inhibitor NXP900 demonstrated a similar trametinib synergy profile to dasatinib, suggesting that SFK inhibition is the likely driver of synergy. ConclusionDasatinib and other SFK inhibitors represent novel candidate treatments for LGSOC and demonstrate synergy with trametinib. Disulfiram represents an additional treatment strategy worthy of investigation.

Vinylpyridine as a Tunable Covalent Warhead Targeting C797 in EGFR.

To further facilitate the discovery of cysteine reactive covalent inhibitors, there is a need to develop new reactive groups beyond the traditional acrylamide-type warheads. Herein we describe the design and synthesis of covalent EGFR inhibitors that use vinylpyridine as the reactive group. Structure-based design identified the quinazoline-containing vinylpyridine 6 as a starting point. Further modifications focused on reducing reactivity resulted in substituted vinyl compound 12, which shows high EGFR potency and good kinase selectivity, as well as significantly reduced reactivity compared to the starting compound 6, confirming that vinylpyridines can be applied as an alternative cysteine reactive warhead with tunable reactivity.

Phosphoproteomic analysis of the adaption of epididymal epithelial cells to corticosterone challenge.

The epididymis has long been of interest owing to its role in promoting the functional maturation of the male germline. More recent evidence has also implicated the epididymis as an important sensory tissue responsible for remodeling of the sperm epigenome, both under physiological conditions and in response to diverse forms of environmental stress. Despite this knowledge, the intricacies of the molecular pathways involved in regulating the adaptation of epididymal tissue to paternal stressors remains to be fully resolved. The overall objective of this study was to investigate the direct impact of corticosterone challenge on a tractable epididymal epithelial cell line (i.e., mECap18 cells), in terms of driving adaptation of the cellular proteome and phosphoproteome signaling networks. The newly developed phosphoproteomic platform EasyPhos coupled with sequencing via an Orbitrap Exploris 480 mass spectrometer, was applied to survey global changes in the mECap18 cell (phospho)proteome resulting from sub-chronic (10-day) corticosterone challenge. The imposed corticosterone exposure regimen elicited relatively subtle modifications of the global mECap18 proteome (i.e., only 73 out of 4171 [∼1.8%] proteins displayed altered abundance). By contrast, ∼15% of the mECap18 phosphoproteome was substantially altered following corticosterone challenge. In silico analysis of the corresponding parent proteins revealed an activation of pathways linked to DNA damage repair and oxidative stress responses as well as a reciprocal inhibition of pathways associated with organismal death. Corticosterone challenge also induced the phosphorylation of several proteins linked to the biogenesis of microRNAs. Accordingly, orthogonal validation strategies confirmed an increase in DNA damage, which was ameliorated upon selective kinase inhibition, and an altered abundance profile of a subset of microRNAs in corticosterone-treated cells. Together, these data confirm that epididymal epithelial cells are reactive to corticosterone challenge, and that their response is tightly coupled to the opposing action of cellular kinases and phosphatases.

Discovery of Novel Bicyclic Pyrazoles as Potent PIP5K1C Inhibitors.

Phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) is generated by phosphatidylinositol 4-phosphate 5-kinases (PIP5Ks) from phosphatidylinositol 4-phosphate (PI4P). Structurally diverse and selective inhibitors against PIP5Ks are required to further elucidate the therapeutic potential for PIP5K inhibition, although the effects of PIP5K inhibition on various diseases and their symptoms, such as cancer and chronic pain, have been reported. Our medicinal chemistry efforts led to novel and potent PIP5K1C inhibitors. Compounds 30 and 33 not only showed potent activity but also demonstrated low total clearance in mice and high levels of kinase selectivity. These compounds might serve as tools to further elucidate the complex biology and therapeutic potential of PIP5K inhibition.

NIBR-LTSi is a selective LATS kinase inhibitor activating YAP signaling and expanding tissue stem cells in vitro and in vivo

The YAP/Hippo pathway is an organ growth and size regulation rheostat safeguarding multiple tissue stem cell compartments. LATS kinases phosphorylate and thereby inactivate YAP, thus representing a potential direct drug target for promoting tissue regeneration. Here, we report the identification and characterization of the selective small-molecule LATS kinase inhibitor NIBR-LTSi. NIBR-LTSi activates YAP signaling, shows good oral bioavailability, and expands organoids derived from several mouse and human tissues. In tissue stem cells, NIBR-LTSi promotes proliferation, maintains stemness, and blocks differentiation invitro and invivo. NIBR-LTSi accelerates liver regeneration following extended hepatectomy in mice. However, increased proliferation and cell dedifferentiation in multiple organs prevent prolonged systemic LATS inhibition, thus limiting potential therapeutic benefit. Together, we report a selective LATS kinase inhibitor agonizing YAP signaling and promoting tissue regeneration invitro and invivo, enabling future research on the regenerative potential of the YAP/Hippo pathway.

Discovery of novel macrocyclic derivatives as potent and selective cyclin-dependent kinase 2 inhibitors

Cyclin-dependent kinase 2 (CDK2) is a member of CDK family of kinases (CDKs) that regulate the cell cycle. Its inopportune or over-activation leads to uncontrolled cell cycle progression and drives numerous types of cancers, especially ovarian, uterine, gastric cancer, as well as those associated with amplified CCNE1 gene. However, developing selective lead compound as CDK2 inhibitors remains challenging owing to similarities in the ATP pockets among different CDKs. Herein, we described the optimization of compound 1, a novel macrocyclic inhibitor targeting CDK2/5/7/9, aiming to discover more selective and metabolically stable lead compound as CDK2 inhibitor. Molecular dynamic (MD) simulations were performed for compound 1 and 9 to gain insights into the improved selectivity against CDK5. Further optimization efforts led to compound 22, exhibiting excellent CDK2 inhibitory activity, good selectivity over other CDKs and potent cellular effects. Based on these characterizations, we propose that compound 22 holds great promise as a potential lead candidate for drug development.

Effects of abrocitinib on pruritus and eczema symptoms and tolerance in patients with moderate‑to‑severe atopic dermatitis in randomized, double‑blind and placebo‑controlled trials: A systematic review and a meta‑analysis.

Abrocitinib is a highly selective Janus kinase 1 (JAK1) inhibitor that can block a multitude of inflammatory signaling pathways that underlie atopic dermatitis (AD). In addition, abrocitinib inhibits JAK1 signaling in sensory neurons to alleviate acute and chronic pruritus during AD. However, substantial variations in efficacy and safety risks remain due to variations in doses applied in clinical use. Therefore for the present study, differences in the efficacy and tolerability of 100 and 200 mg abrocitinib for treating pruritus and eczema symptoms in patients with moderate-to-severe AD were evaluated compared with placebo. Specifically, randomized controlled trials (RCTs) of abrocitinib compared with placebo for the treatment of moderate-to-severe AD were searched on Pubmed, E.B. Stephens Company, China National Knowledge Infrastructure, Wanfang Medical network, Web of Science and related Clinical Trials Registry up to November 2023. In total, two researchers evaluated the quality of the included literature according to the Cochrane Handbook of Systematic Reviews. RevMan 5.3 software was used to conduct a meta-analysis of the efficacy and safety indicators in a cross-comparison of the effects exerted by placebo and 100 and 200 mg abrocitinib. A total of 1,825 patients with moderate-to-severe AD were included across five double-blind, placebo RCTs. Compared with the placebo group, during the double-blind trial period, significant improvements were observed in the investigator's global assessment score, response rate of eczema area and severity index (EASI)-50, EASI-75, EASI-90 and pruritus numerical rating scale (P-NRS) in the 100 and 200 mg abrocitinib groups (P<0.05). However, pairwise control analysis of the 100 and 200 mg group yielded significant differences (P<0.05) in all of the aforementioned therapeutic indicators except for the P-NRS score. In terms of safety, compared with the placebo group, there were significantly higher incidence of nausea, upper respiratory tract viral infection, infections and infestations in the 100 mg abrocitinib group (P<0.05). In addition, there were significantly higher incidence of nausea, gastrointestinal disorder, headache and dizziness in the 200 mg group (P<0.05). There were also significant differences in the incidence of nausea, gastrointestinal disorder and dizziness between the 100 and 200 mg groups (P<0.05). For patients with moderate-to-severe AD, oral administration of 100 or 200 mg abrocitinib once/day was concluded to ameliorate skin pruritus and eczema symptoms to varying degrees, with the efficacy significantly superior at the 200 mg dose. However, the risk of a number of adverse reactions, such as headache, dizziness, nausea and gastrointestinal dysfunction, is also significantly increased. Therefore, patients should be made aware of the risk of adverse drug effects prior to the administration of long-term high abrocitinib doses. Furthermore, large-scale, multi-center, rigorous clinical trials remain necessary to validate the findings from the present study.

Peficitinib alleviated acute lung injury by blocking glycolysis through JAK3/STAT3 pathway

Peficitinib is a selective Janus kinase (JAK3) inhibitor recently developed and approved for the treatment of rheumatoid arthritis in Japan. Glycolysis in macrophages could induce NOD-like receptor (NLR) family and pyrin domain-containing protein 3 (NLRP3) inflammasome activation, thus resulting in pyroptosis and acute lung injury (ALI). The aim of our study was to investigate whether Peficitinib could alleviate lipopolysaccharide (LPS)-induced ALI by inhibiting NLRP3 inflammasome activation. Wild type C57BL/6J mice were intraperitoneally injected with Peficitinib (5 or 10 mg·kg−1·day−1) for 7 consecutive days before LPS injection. The results showed that Peficitinib pretreatment significantly relieved LPS-induced pulmonary edema, inflammation, and apoptosis. NLRP3 inflammasome and glycolysis in murine lung tissues challenged with LPS were also blocked by Peficitinib. Furthermore, we found that the activation of JAK3/signal transducer and activator of transcription 3 (STAT3) was also suppressed by Peficitinib in mice with ALI. However, in Jak3 knockout mice, Peficitinib did not show obvious protective effects after LPS injection. In vitro experiments further showed that Jak3 overexpression completely abolished Peficitinib-elicited inhibitory effects on pyroptosis and glycolysis in LPS-induced RAW264.7 macrophages. Finally, we unveiled that LPS-induced activation of JAK3/STAT3 was mediated by toll-like receptor 4 (TLR4) in RAW264.7 macrophages. Collectively, our study proved that Peficitinib could protect against ALI by blocking JAK3-mediated glycolysis and pyroptosis in macrophages, which may serve as a promising candidate against ALI in the future.

Early and Sustained Improvements in Symptoms and Quality of Life with Upadacitinib in Adults and Adolescents with Moderate-to-Severe Atopic Dermatitis: 52-Week Results from Two Phase III Randomized Clinical Trials (Measure Up 1 and MeasureUp2).

Atopic dermatitis is a chronic inflammatory disease characterized by increased itch, skin pain, poor sleep quality, and other symptoms that negatively affect patient quality of life. Upadacitinib, an oral selective Janus kinase (JAK) inhibitor with greater inhibitory potency for JAK1 than JAK2, JAK3, or tyrosine kinase 2, is approved to treat moderate-to-severe atopic dermatitis. We aimed to evaluate the effect of upadacitinib on patient-reported outcomes over 52weeks in adults and adolescents with moderate-to-severe atopic dermatitis. Data from two phase III monotherapy trials of upadacitinib (Measure Up 1, NCT03569293; Measure Up 2, NCT03607422) were integrated. Changes in pruritus, pain, other skin symptoms, sleep, quality of life, mental health, and patient impression were evaluated. Patient-reported outcome assessments included the Worst Pruritus Numerical Rating Scale, Patient-Oriented Eczema Measure, Dermatology Life Quality Index, Atopic Dermatitis Symptom Scale, Atopic Dermatitis Impact Scale, Hospital Anxiety and Depression Scale, SCORing Atopic Dermatitis index, Patient Global Impression of Severity, Patient Global Impression of Change, and Patient Global Impression of Treatment. Minimal clinically important differences, achievement of scores representing minimal disease burden, and the change from baseline were evaluated in patients who received upadacitinib through week 52 and in patients who received placebo through week16. This analysis included 1609 patients (upadacitinib 15 mg, N=557; upadacitinib 30 mg, N=567; placebo, N=485). Baseline demographics and disease characteristics were generally similar across all arms. The proportion of patients treated with upadacitinib reporting improvements in itch increased rapidly by week 1, increased steadily through week 8, and was sustained through week 52. Patients receiving upadacitinib also experienced improvements in pain and other skin symptoms by week 1, which continued through week 16; improvements were maintained through week 52. Patient reports of improved sleep increased rapidly from baseline to week 1, increased steadily through week 32, and were sustained through week 52. Patients experienced quality-of-life improvements through week 8, which were maintained through week 52. By week 1, patients in both upadacitinib groups experienced rapid improvements in emotional state, and by week 12, patients also achieved meaningful improvements in anxiety and depression. Improvements in mental health continued steadily through week 32 and were maintained through week 52. Patients treated with upadacitinib30mg generally experienced improvements in patient-reported outcomes earlier than those treated with upadacitinib 15 mg. Through week 16, patients receiving upadacitinib experienced greater improvements versus those receiving placebo in all assessed patient-reported outcomes. Adults and adolescents with moderate-to-severe atopic dermatitis treated with once-daily upadacitinib 15 or 30 mg experienced early improvements in itch, pain, other skin symptoms, sleep, quality of life, and mental health that were sustained through week52. ClinicalTrials.gov identifiers NCT03569293 (13 August 2018) and NCT03607422 (27 July 2018).

Discovery of pyrido[3,2-d]pyrimidin-6(5H)-one derivatives as checkpoint kinase 1 (CHK1) inhibitors with potent antitumor efficacy

Checkpoint kinase 1 (CHK1) plays a crucial role in the DNA damage response pathway, making it an attractive target for cancer therapy. Herein, we present the synthesis, optimization, and evaluation of selective CHK1 inhibitors with a pyrido[3,2-d]pyrimidin-6(5H)-one scaffold. Among them, compound 11 showed single-digit nanomolar potency against CHK1 (IC50: 0.55 nM) with good kinase selectivity. Notably, 11 showed anti-proliferative effect in MV-4-11 cells singly (IC50 = 202 nM) and a synergistic effect in combination with gemcitabine in HT-29 cells (IC50 = 63.53 nM). Furthermore, the combination of 11 and gemcitabine exhibited synergistic effect in the HT-29 xenograft mouse model. Overall, this work provides a strong foundation for the development of selective CHK1 inhibitors and the therapeutic strategy for cancer.

Abstract 5966: PRT2527, a novel highly selective cyclin-dependent kinase 9 (CDK9) inhibitor, has potent antitumor activity in combination with BTK and BCL2 inhibition in various lymphoid malignancies

Abstract CDK9 is a master regulator of transcription that modulates transcription elongation via phosphorylation of RNA polymerase II. Short-term inhibition of CDK9 depletes short-lived transcripts and labile proteins such as MCL1, BFL1 and MYC to promote cancer cell death. We previously described PRT2527, a novel, potent, highly selective CDK9 inhibitor with anti-leukemic activity in various preclinical models. PRT2527 is currently under evaluation in a Phase I clinical trial in patients with relapsed/refractory hematologic malignancies (NCT05159518). Here we demonstrate PRT2527 potently inhibits cancer cell growth and induces cell death in various models of Diffuse Large B Cell Lymphoma (DLBCL), Mantle Cell Lymphoma (MCL) and Chronic Lymphocytic Leukemia (CLL), through depletion of MCL1, BFL1 and MYC. Specifically, we show that brief treatment with PRT2527 (6h) potently induces apoptosis and inhibits proliferation in DLBCL, MCL and CLL cell lines. We also show that the addition of BTK inhibitors (BTKi) or BCL2 inhibitors (BCL2i) potentiates the apoptosis response induced by PRT2527 in vitro and leads to more robust and durable responses including complete tumor regressions in DLBCL CDX and PDX models in vivo. In all studies, the combination of PRT2527 with BTKi or BCL2i is well tolerated. Mechanistically, we show that BTK inhibition upregulates BMF, an endogenous inhibitor of BCL2 and BCL-xL, driving cells towards MCL1 and BFL1 dependency in TMD-8 cells. Concurrent depletion of MCL1 and BFL1 with PRT2527 leads to complete inhibition of Bcl-2- family mediated survival signaling, potently inducing cell death. In similar fashion, Venetoclax-driven BCL2 inhibition also potentiates the anti-tumor activity of PRT2527. Consistent with these findings in vitro, we observe reduced BFL1 and MCL1 as well as increased BMF in tumors of mice treated with BTKi and PRT2527 suggesting complete Bcl-2 family inhibition. We also demonstrate potent induction of apoptosis in peripheral blood mononuclear cells isolated from patients with both treatment-naïve and relapse/refractory CLL following brief treatment with PRT2527 (6h). Co-treatment with BTKi or BCL2i further enhances the apoptotic effect of PRT2527, similarly suggesting combinatorial benefit. Additionally, we characterize the activity of PRT2527 in models of BTKi relapsed/refractory MCL where increased CDK9 dependency has previously been described. In an Ibrutinib-resistant Mino CDX model, we observe increased tumor growth inhibition following treatment with PRT2527 compared to tumors derived from parental Mino cells, suggesting PRT2527 is efficacious in BTKi-resistant MCL. Altogether these data provide a rationale for evaluating PRT2527 in combination with BTK and BCL2 inhibitors for the treatment of patients with relapsed/refractory lymphoid malignancies. Citation Format: Norman Fultang, Ashley M. Schwab, Sophia McAneny-Droz, Diane Heiser, Peggy Scherle, Neha Bhagwat. PRT2527, a novel highly selective cyclin-dependent kinase 9 (CDK9) inhibitor, has potent antitumor activity in combination with BTK and BCL2 inhibition in various lymphoid malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5966.

Abstract 6500: Dependence of EGFR-mutant NSCLC on MET as demonstrated by vebreltinib, a novel and selective brain-penetrating MET kinase inhibitor

Abstract Background: Although EGFR inhibitors (EGFRi), preferably osimertinib, have offered substantial benefit for the classical plus T790M EGFR-mutant NSCLC patients, drug resistance develops and poses a critical challenge for long-term survival. The resistance is rooted to either on-target secondary EGFR mutations or compensatory oncogenic pathways bypassing EGFRi intervention, one of which appears to be treatment-acquired MET amplification (METamp). In this study we set out to understand the conditions when the tumor cells become dependent on MET. Methods: NSCLC patient-derived tumor models (PDX) carrying classical and T790M EGFR driver mutations were selected based on EGFRi history and concurrent MET gene copy numbers or RNA expression levels: Type 1) resistant to osimertinib with METamp; Type 2) partially resistant to osimertinib with METamp; and Type 3) sensitive to osimertinib with low MET expression, no METamp. These PDX xenograft mice were treated with vebreltinib (APL-101) at clinically relevant doses as single agent or in combination with osimertinib. The pharmacodynamic effects on EGFR and MET phosphor (P)- and total proteins were analyzed from tumor samples harvested during and at the end of treatments. Results: Vebreltinib single agent inhibited tumor growth of the type-1 PDX. One such PDX was resistant to chemotherapies, BRAF inhibitor and EGFRi including osimertinib, was inhibited by vebreltinib with ED50 of 3 mg/kg (p.o/q.d.). Another such PDX tumor appeared to be eradicated by vebreltinib treatment at 10 mg/kg (p.o/q.d.) and the mice became tumor-free on Day 18 and remained so after dosing stop on day 21 with last observation at study end (Day 70). In comparison, reference METi savolitinib showed only partial tumor size reduction against the same PDX tumor, not as durable as vebreltinib, and did not eradicate tumor growth at the same dose as vebreltinib or in combo with Osimertinib. Re-grown tumor after savolitinib osimertinib combo dosing stop was inhibited completely by vebreltinib follow-on treatment. In the type-2 PDX, tumor growth was partially inhibited by vebreltinib but was completely inhibited by combo of vebreltinib and osimertinib. The anti-tumor activity of vebreltinib in type-1 and type-2 PDX was associated with pharmacodynamic inhibition of P-MET and P-EGFR and MET protein degradation. In the type-3 PDX, tumor growth appeared to be eradicated by osimertinib and not inhibited by vebreltinib. However, addition of vebreltinib suppressed tumor re-growth after osimertinib dosing stop. Conclusion: EGFR-mutant NSCLC resistance to EGFRi may be dependent on MET pathway regardless of acquired METamp, whereas METamp may dictate the degree of MET dependence. Our data supports the hypothesis that adding vebreltinib to EGFRi overcome MET dependent resistance with durable effect or preventing MET dependent resistance to maximize therapeutic benefits. Citation Format: Xiaoling Zhang, Elaine Liu, Yan Song, Peony Yu, Sanjeev Redkar, Guo-Liang Yu. Dependence of EGFR-mutant NSCLC on MET as demonstrated by vebreltinib, a novel and selective brain-penetrating MET kinase inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6500.

Abstract 1816: In silico design of novel multitarget small molecule inhibitor LCI139 for the treatment of PTEN-mutant endometrial carcinoma

Abstract Background: Previously we described multitarget CDK4/6-CDK9 inhibitor, LCI133 for the treatment of endometrial carcinoma (EC). We have optimized LCI133’s chemical properties to increase PI3K p110α potency to better target epithelial-derived cancers, resulting in nanomolar potent LCI139. We characterized LCI139’s in vitro potency, target specificity, activity in in vitro EC models, safety against normal cell lines. Methods: LCI139’s chemical structure was determined using nuclear magnetic resonance and high-resolution mass spectrometry. Specific target inhibition was assessed by cell-free kinase hotspot/lipid kinase assay and selectivity profile. LCI139 (0.001-10μM) was screened against EC cell lines AN3CA, RL95-2, Ishikawa, KLE, HEC-1-A and -B for 48 hours; IC50 values determined by CellTiter-Glo Assay. EC cells were treated with LCI139 or single agent control for 24 hours and stained with Annexin V/7-AAD to assess apoptosis by cytometer. Representative PTEN wildtype (HEC-1) and mutant (AN3CA) EC cell lines were chosen for mechanistic studies and treated 6 to 24 hours with multitarget or single agent inhibitor. RNA was isolated to determine gene expression by qRT-PCR, cells were stained with Vybrant DyeCycle Violet to determine cell cycle arrest by cytometer, or cellular lysates were prepared for Western blot (WB). Results: LCI139’s has a molecular weight of 502.57Da and is a highly potent PI3Kα-CDK4/6-CDK9 inhibitor by cell-free kinase assays: 0.070µM (PI3Kα), 0.461µM (PI3Kγ), 0.214µM (PI3Kδ), 0.0015µM (CDK4), 0.0036µM (CDK6), 0.000039µM (CDK9). KINOMEscan revealed a favorable S(35) selectivity score of 0.221 screened against 403 non-mutant kinases. PTEN mutant AN3CA (IC50 243.6nM), RL95-2 (IC50 22.8nM), Ishikawa (IC50 9.8nM) had nanomolar LCI139 IC50 values, while PTEN-wild type cell lines were insensitive up to 10μM; confirmed by dose-dependent apoptosis and colony-forming assays. WB revealed LCI139 treatment ablated MCL-1 expression, decreased phosphorylation of AKT, Rb, Rpb1 (Ser2), induced significant amounts of cPARP. MCL-1 ablation correlated to decreased Mcl1 expression (p&amp;lt;0.0010) assessed 6 hours post treatment. LCI139 generated heterogenous cell line specific effects on cell cycle progression and arrest. LCI139 displayed a favorable toxicity profile against normal human kidney cells (HEK-293) compared to single agent controls. Conclusions: Triple PI3K-CDK4/6-CDK9 inhibitor, LCI139, is nanomolar potent against PTEN mutant EC cell lines and displays decreased toxicity compared with CDK9 inhibitor reference compounds, AZD4573 and flavopiridol. Our results merit further mechanistic dissection of PTEN interaction with PI3K pathway signaling, cell cycle machinery, CDK9-mediated transcriptional control. These data support extensive in vivo potency, toxicity, PK/PD studies to assess LCI139’s clinical utility in treating EC. Citation Format: Vaidehi Mujumdar, Ritchie Delara, Hailey Dryden, Dhananjaya Pal, Krishnaiah Maddeboina, Page Mangum Arditti, Bharath Yada, Robert Wendel Naumann, Yovanni Casablanca, Erin Crane, Jubilee Brown, Donald Durden, Cody McHale. In silico design of novel multitarget small molecule inhibitor LCI139 for the treatment of PTEN-mutant endometrial carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1816.

Abstract 4540: SY-7021, a novel DNA-PK inhibitor, exhibits significant anti-tumor activity in vitro and in vivo

Abstract Cancer therapies such as ionizing radiation or topoisomerase II inhibitors (for example, doxorubicin) induce DNA double-strand breaks (DSBs), which can subsequently be repaired by homologous recombination or non-homologous end joining (NHEJ). DNA-dependent protein kinase (DNA-PK), a nuclear serine/threonine protein kinase complex, is a pivotal component of the NHEJ process that maintain genome integrity. The critical role of DNA-PK in DNA damage response (DDR) and the dysregulated DNA-PK expression in cancer make it an intriguing therapeutic target for cancer, especially when combined with DSBs agents. Here, we identified a highly potent and selective DNA-PK inhibitor, SY-7021, with an IC50 value of 0.242 nM on DNA-PK kinase and high selectivity on ATM and ATR (greater than 400-fold) in biochemical assays. In a reporter assay for NHEJ repair, SY-7021 was able to dose-dependently reduce cellular NHEJ efficiency. Functionally, SY-7021 inhibited cell proliferation in multiple cancer cell lines alone or combined with doxorubicin. Basically, combination of SY-7021 and doxorubicin induced significant G2/M phase arrest and cell apoptosis, as well as enhanced the phosphorylation on Ser139 of γH2AX, Tyrosine 68 of CHK2 and Serine15 of p53 in MDA-MB-468 cells. In a subcutaneous NCI-H1703 xenograft tumor model, SY-7021 administered orally twice daily achieved dose-dependent tumor growth inhibition in vivo, with a tumor growth inhibition (TGI) of 105.6% at 60 mg/kg and no significant weight loss was observed. In addition, SY-7021 demonstrates good PK properties and acceptable safety profiles in in vivo studies. Collectively, SY-7021, a potent and selective DNA-PK inhibitor, shows significant inhibition on tumor growth in in vitro and in vivo studies, providing a rationale treatment for multiple tumors in monotherapy or in combination with other agents. Citation Format: Kai Zhang, Zhihua Liu, Yuhao Gao, Chang Lu, Shikang Cheng, Xijie Liu, Hong Luo, Yinghui Sun. SY-7021, a novel DNA-PK inhibitor, exhibits significant anti-tumor activity in vitro and in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4540.

Abstract 3631: Preclinical and clinical pharmacodynamic characterization of BBI-355, a novel, orally bioavailable, and selective CHK1 inhibitor being evaluated in the first-in-human Phase 1/2 POTENTIATE clinical trial of patients with cancer harboring oncogene amplifications

Abstract Pharmacodynamic (PD) biomarkers play an integral role in the rational development of novel therapeutic agents. Critically, PD biomarkers enable the measurement and confirmation of a drug’s intended effect in early clinical trials. Integration of PD biomarker assessments in early therapeutic development can inform the relationship between drug dose and regimen, target modulation, and biological tumor response, which are fundamental to determining the efficacy and safety of novel therapeutic agents. Boundless Bio is evaluating a novel, orally available, and selective Checkpoint Kinase 1 (CHK1) inhibitor, BBI-355, in the Phase 1/2 clinical trial, POTENTIATE (NCT05827614), in patients with solid tumors harboring oncogene amplification including those on extrachromosomal DNA (ecDNA). ecDNA-enabled oncogene amplification endows cancers with significant genomic plasticity, which can facilitate resistance to targeted therapy (Nathanson et al., 2014), and which is also associated with significantly shorter survival (Kim et al., 2020). While ecDNA provides a fitness advantage to cancer cells, its unique features also confer certain liabilities, including elevated DNA replication stress (RS). Elevated RS results in DNA damage and makes ecDNA+ cancers hyper-reliant on the master regulator of RS, CHK1, which manages DNA replication and repair to maintain cell viability (Chowdhry, 2022). Due to their elevated RS and hyper-reliance on CHK1, ecDNA+ cancer cells are rendered more susceptible to CHK1 inhibition than cancer cells that lack ecDNA. Prior clinical CHK1 inhibitor programs have lacked effective clinical PD biomarker assays. Here, we present preclinical data supporting the use of phosphorylated-CHK1 Ser345 (pCHK1-S345), a proximal marker of RS, as a PD biomarker for CHK1 inhibition by BBI-355. In preclinical studies, BBI-355 treatment induced pCHK1-S345 protein expression by immunoblotting in tumor cell lines in vitro and in xenograft tumors in vivo, which corresponded with tumor cell cytotoxicity. We went on to demonstrate in rats the utility of skin as a tumor surrogate tissue to detect CHK1 inhibitor PD effect through induction of pCHK1-S345 expression by immunohistochemistry. Finally, and for the first time, PD activity of a CHK1 inhibitor in humans was demonstrated by increased pCHK1-S345 expression by immunohistochemistry in skin biopsies from patients enrolled on the POTENTIATE study and treated with BBI-355. In conclusion, pCHK1-S345 is a useful PD biomarker for confirming clinical on-target activity of BBI-355 and may be useful to inform the pharmacologically active range of BBI-355 to support clinical development. Citation Format: Debbie Liao, Ryan Hansen, Sudhir Chowdhry, Evan Holmes, Julie Wiese, Auzon Steffy, AnneMarie Pferdekamper, Edison Tse, Salvador Garcia, Rebecca Reynolds, Sara Weymer, Klaus Wagner, Chris Hassig, Shailaja Kasibhatla. Preclinical and clinical pharmacodynamic characterization of BBI-355, a novel, orally bioavailable, and selective CHK1 inhibitor being evaluated in the first-in-human Phase 1/2 POTENTIATE clinical trial of patients with cancer harboring oncogene amplifications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3631.

Abstract 2031: A phase 1b/2 clinical study of onvansertib in combination with FOLFIRI/bevacizumab revealed a new role of PLK1 in regulating the hypoxia pathway in KRAS-mutant colorectal cancer

Abstract Background: Onvansertib (Onv) is an oral, small molecule, selective PLK1 kinase inhibitor that demonstrated clinical activity at tolerated drug exposures in combination with FOLFIRI/Bevacizumab (Bev) in the 2nd-line treatment of mutated KRAS (mKRAS) metastatic colorectal cancer (mCRC) (Lenz, JCO, 2022). Here we explored biomarkers of response to the combination therapy and their associated biology. Methods: In a Phase 1b/2 study, mKRAS mCRC patients with prior exposure to oxaliplatin (with or without Bev) were treated with Onv (Days 1-5 and 15-19) in combination with FOLFIRI/Bev (Days 1 and 15) of each 28-day cycle (NCT03829410). Efficacy endpoints included objective response rate (ORR, RECIST v1.1), progression-free survival (PFS) and duration of response (DoR). Preclinically, the combination of Onv and Bev was tested in 3 mKRAS CRC xenograft models. The role of PLK1 in hypoxia was assessed through protein and RNA analyses of mKRAS CRC cell lines exposed to low oxygen levels and treated with Onv. Results: As of 25-AUG-2023, 66 patients were evaluable for efficacy in the Phase 1b/2 and ORR of Onv + FOLFIRI/Bev was 28.8%. Patients had a median PFS of 9.8 months [95%CI: 7.6, 12.6] and median DoR of 11.7 months [95%CI: 9.0, NR]. An analysis of baseline characteristics revealed that patients not exposed to Bev in the 1st-line setting (Bev-naïve patients, n=15) achieved superior clinical benefit compared to Bev-exposed patients (n=51). Bev-naïve patients had significantly higher ORR (73.3% versus 15.7%, p&amp;lt;0.0001) and longer median PFS (14.9 months [95%CI: 10.5, NR] versus 7.8 months [95%CI: 5.6, 9.8], p=0.0013) than Bev-exposed patients. This data prompted us to assess the effect of Onv+Bev in mKRAS CRC xenograft models. Onv+Bev significantly reduced tumor growth in the 3 xenograft models tested. The combination resulted in a greater decrease in tumor vascularization and tumor cell proliferation, and a corresponding increase in apoptosis, compared to either monotherapy. Further, the effect of Onv on the hypoxia pathway was assessed in mKRAS CRC cell lines. Our findings indicated that under low oxygen levels, Onv inhibited the hypoxia pathway by reducing the expression of the hypoxia-inducible factor 1-alpha (HIF1α) protein, and the mRNA expression of its downstream targets. Conclusions: Onv + FOLFIRI/Bev resulted in greater clinical benefit in Bev- naïve than Bev-exposed mKRAS CRC patients. Preclinical studies revealed synergistic effect of Onv and Bev in mKRAS CRC xenografts, and a new function of PLK1 in modulating the hypoxia pathway through the regulation of HIF1α. Collectively, this data supports a crosstalk between PLK1 and angiogenesis, and further exploration of Onv + FOLFIRI/Bev therapy in Bev-naïve mCRC patients with KRAS mutation. Citation Format: Maya Ridinger, Anju Karki, Ramanand A. Subramanian, Errin Samuelsz, Divora Yemane, Roy Kim, Chu-Chiao Wu, Fairooz F. Kabbinavar, Tod Smeal, Heinz-Josef Lenz. A phase 1b/2 clinical study of onvansertib in combination with FOLFIRI/bevacizumab revealed a new role of PLK1 in regulating the hypoxia pathway in KRAS-mutant colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2031.

Abstract 597: Vebreltinib: A novel brain-penetrating MET kinase inhibitor demonstrates the mechanism of action and pharmacological anti-tumor activity in diverse patient-derived MET-dysregulated tumor models at clinically relevant drug levels

Abstract Background: Dysregulation of the MET signaling pathway drives oncogenesis in many human cancers, the majority of which have not been addressed by a MET targeted therapy albeit the emerging therapies for MET exon 14 skipping (MET ex14) NSCLC. We have developed a novel highly selective MET kinase inhibitor vebreltinib (APL-101, PLB1001, bozitinib) to address unmet medical need. Vebreltinib is being jointly investigated in ongoing pivotal Phase 2 and 2/3 studies in MET-dysregulated NSCLC, primary CNS cancers and basket solid tumors in China (as PLB-1001) and globally ex-China (as APL-101). The Phase 1 portion of both studies have been concluded with the same RP2D (200 mg BID by oral administration). Vebreltinib demonstrated blood-brain barrier permeability in PLB-1001 Phase 1 glioblastoma study as previously reported. Methods: MET enzyme kinetics and vebreltinib intrinsic potency were studied by a radiometric MET kinase assay. Selectivity was profiled by in vitro KINOME. Vebreltinib cellular activity on MET signaling and in vivo anti-tumor activity were assessed in tumor cell lines and patient-derived tumors (PDX) carrying MET alterations. Results: Vebreltinib is an ATP-competitive inhibitor with Ki of approximately 2.2 nM for recombinant MET kinase and inhibited the native form of activated MET with 0.52 nM IC50. Vebreltinib demonstrated exquisite selectivity in KINOMEscan selectivity panel. Vebreltinib inhibited HGF-dependent and -independent MET signaling in KP4 tumor cell lines with nanomolar concentrations. Vebreltinib inhibited IL-3 independent Ba/F3 cell lines carrying exogenous MET ex14 or with secondary mutations induced by other MET TKIs. Vebreltinib inhibited MKN45 gastric tumor xenograft in vivo with ED90 of 6 mg/kg PO/QD. Furthermore, Vebreltinib demonstrated strong anti-tumor activity in a variety of PDX tumor types carrying MET ex14, MET fusions, and MET amplification. In these preclinical studies, the total and unbound vebreltinib plasma concentration observed at steady state trough of RP2D was used as a clinically relevant drug level to interpret Vebreltinib efficacy in blocking oncogenic MET signaling, proliferation of Ba/F3 cell lines with a broad spectrum of MET mutations and in vivo tumor growth in PDXs carrying diverse MET alterations. We demonstrated the rationale for Vebreltinib to be an efficacious single-agent MET inhibitor in treating tumors carrying MET driver alterations beyond MET ex14 NSCLC. Conclusion: Vebreltinib is a novel potent MET kinase inhibitor showing promising preclinical activity against PDXs from diverse organs sites and genomic alterations such as MET ex14, MET fusion, MET amplification, or HGF over-expression at clinically relevant drug levels, providing proof-of-concepts for continued clinical development. Citation Format: Xiaoling Zhang, Elaine Liu, Yan Song, Peony Yu, Sanjeev Redkar, Guo-Liang Yu. Vebreltinib: A novel brain-penetrating MET kinase inhibitor demonstrates the mechanism of action and pharmacological anti-tumor activity in diverse patient-derived MET-dysregulated tumor models at clinically relevant drug levels [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 597.